CN114314860A

CN114314860A - Method for rapidly converting bisphenol A in water body

Info

Publication number: CN114314860A
Application number: CN202210026238.6A
Authority: CN
Inventors: 孙凯; 刘婕; 齐学敏; 司友斌
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-04-12

Abstract

The invention belongs to the cross-fusion field of fungus biology, enzymatic reaction kinetics, environmental pollution restoration science and the like, and particularly relates to a method for rapidly converting bisphenol A in a water body, which comprises the steps of adding a bisphenol A-containing water body with a preservation number of CGMCC No: 5.2199 fermenting coarse laccase liquid or mycelium homogenate of Trametes hirsute La-7, which can simply, rapidly and efficiently treat bisphenol A polluted water body.

Description

Method for rapidly converting bisphenol A in water body

Technical Field

The invention belongs to the cross-fusion fields of fungus biology, enzymatic reaction kinetics, environmental pollution restoration and the like, and particularly relates to preparation of trametes hirsutella rough laccase liquid and mycelium homogenate and application thereof in bisphenol A conversion.

Background

Environmental hormones can destroy the normal endocrine function of organisms, thereby disturbing the reproduction of wild species and threatening the health and survival of human beings. Bisphenol a is widely used as a synthetic raw material of high polymer substances such as epoxy resin, polycarbonate and the like to manufacture plastic products. The compounds have typical environmental hormonal effects and enter animals and humans through bioaccumulation even at low dose exposure, damage the liver, cause hormonal imbalances, affect reproductive development, and even induce cancer. Currently, researchers have detected high concentrations of bisphenol a in rivers, groundwater, animals, and humans. This is because the conventional water treatment process cannot effectively remove bisphenol a in the water body and may generate harmful by-products; the adsorption, permeation, membrane separation and other technologies can realize phase interface transfer of bisphenol A, but the subsequent treatment cost is higher; microbial metabolism has the advantages of low cost, environmental protection and the like, but the degradation period is longer, the microbial metabolism is very easily influenced by indigenous microorganisms, and high-concentration bisphenol A can inhibit or kill bacterial strains. In recent years, the emerging technologies of photocatalysis, deep oxidation, electrochemistry and the like are widely concerned in the rapid conversion and elimination of bisphenol A, and the disadvantages of the technologies are high energy consumption, high processing price and residual risk of toxic byproducts.

Currently, the biodegradation and conversion of bisphenol A by fungal extracellular enzymes (e.g., ligninolytic enzymes, peroxidases, and oxidases) is highly appreciated by researchers. Extracellular laccase (EC 1.10.3.2) secreted by white-rot fungi (white-rot fungi) belong to a copper-containing polyphenol oxidoreductase which is ubiquitous in nature. The catalytic active site of the enzyme is formed by 4 Cu ions together, wherein a T1-Cu site is responsible for oxidizing a substrate molecule into an active intermediate, and a trinuclear copper cluster center (formed by 1T 2-Cu site and 2T 3-Cu sites together) is responsible for reducing dissolved oxygen in water into water. Fungal laccases have been shown to initiate one-electron oxidative migration of various phenolic contaminants such as bisphenol-A with molecular oxygen as the final electron acceptor, and to produce only water as the sole by-product. The laccase can generate unstable active free radicals or quinone intermediates in the process of initiating the bisphenol A oxidation, and the unstable active free radicals or quinone intermediates can attack parent compounds to oxidize and decompose the parent compounds into small molecular products and can generate macromolecular polymerization products through the covalent bonding effect promoted by free radicals. Nevertheless, the separation and purification cost of fungal laccase is high, so that the fungal laccase is difficult to be applied to bisphenol A sewage treatment on a large scale. In view of the oxidative decomposition and free radical combination characteristics of fungal laccase, can simply, rapidly and efficiently remove bisphenol a in water by using hypha homogenate prepared from extracellular crude laccase solution and mycelium thereof secreted by laccase-producing fungi separated and screened in a specific environment matrix? The creative idea needs to be broad.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to simply, rapidly and efficiently convert bisphenol A in water.

The technical scheme of the invention is as follows: a method for rapidly converting bisphenol A in a water body is characterized in that a water body containing bisphenol A is added with a preservation number of CGMCC No: 5.2199 fermented crude laccase liquid or mycelium homogenate of Trametes hirsute La-7.

Further, Mn is added when the fermentation crude laccase liquid is added²⁺Preferably, Mn²⁺The concentration was 0.5 mM.

Further, the fermented laccase crude liquid and mycelium homogenate are prepared by the following method: the preservation number is CGMCC No: 5.2199 Trametes hirsutella (Trametes hirsute La-7) is inoculated into liquid medium, and the mixture is protected from light at 28-32 ℃ and 120-180 r.min^-1Fermenting and incubating for 3-6d, performing solid-liquid separation, wherein the supernatant is fermented crude laccase liquid, and mycelium is uniformly ground to obtain mycelium homogenate.

Preferably, the fermentation incubation parameter is 30 ℃, 150 r.min^-1Liquid fermentation incubation 4 d.

The inventor separates and screens a laccase-producing trametes hirsuta (T.hirsute La-7) from surface soil of a drain outlet of a certain pig farm in an earlier stage, wherein the preservation number of the strain is CGMCC No: 5.2199. the fungus can be used for degrading 17 beta-estradiol in water body. When the strain is used for degrading bisphenol A in a water body, the low-dose bisphenol A can be effectively converted by the laccase-producing fungus, and the high-dose bisphenol A can completely inhibit the growth and propagation of the fungus. However, this inhibition was completely removed by the laccase crude liquid and hyphal homogenate of the strain.

Compared with the prior art, the invention has the following beneficial effects:

the biotransformation rates of tramete hirsute (T.hirsute La-7) crude laccase liquid and mycelium homogenate liquid to bisphenol A in a water body within 6 hours are respectively 80.6% -90.7% and 58.1% -84.2%. Compared with the prior methods of advanced oxidation, bacterial degradation, fungal metabolism and the like, the homogenate of the trametes hirsuta laccase liquid and the hyphae obviously shortens the biotransformation period of the bisphenol A. The process can not only induce bisphenol A to be oxidized and cracked to form 4-isopropyl phenol, phenol and other decomposition products with low molecular weight and low toxicity, but also induce bisphenol A free radical covalent bonding to generate dimer, trimer and other polymerization products with high molecular weight and stable structure.

The trametes hirsuta crude laccase liquid and mycelium homogenate prepared by the invention can be used for simply, quickly and efficiently treating bisphenol A polluted water body. The biological treatment process has the advantages of simple preparation process, low production cost, controllable operation flow, short conversion period and the like, and accords with the development concepts of green, economy, energy conservation and environmental protection. The high resolution mass spectrometry used¹³The C stable isotope ratio method can quickly and accurately identify bisphenol A conversion intermediate products. Therefore, the trametes hirsuta crude laccase liquid and mycelium homogenate liquid are beneficial to research and development of a novel microbial agent, and have great application potential and wide commercial prospect in aspects of water environment treatment, resource recycling and the like.

Drawings

FIG. 1 bioconversion of trametes hirsuta with different concentrations of bisphenol A, (a) bioconversion with 0.25mM bisphenol A, (b) bioconversion with 0.5mM bisphenol A, (c) bioconversion with 1.0mM bisphenol A;

FIG. 2 is a process for preparing a homogenate of trametes hirsutus laccase solution and mycelia;

FIG. 3 bioconversion of trametes hirsuta laccase liquid to bisphenol A, (a) adding the laccase liquid only, (b) adding the laccase liquid and Mn simultaneously²⁺；

FIG. 4 bioconversion of mycelium and mycelium homogenates to bisphenol A, (a) is mycelium and (b) is mycelium homogenate.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Trametes hirsuta La-7 is separated from surface soil of a drain outlet of a certain pig farm, and is preserved in China general microbiological culture collection center on 27 th month 09 in 2019, with the culture preservation number of CGMCC No: 5.2199, deposit address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North China, has been published in CN 110846231A.

Example 1 bioconversion Effect of trametes hirsuta on bisphenol A

The influence of trametes hirsutus (T.hirsute La-7) on the biotransformation of bisphenol A is studied by adopting a microbial fermentation culture method. The operation steps are briefly described as follows: (1) inoculating trametes hirsuta mycelium in a solid laccase-producing medium, and standing and incubating for 7d at 30 ℃ in the dark to obtain a trametes hirsuta cake with the diameter of 6.9 cm; each liter of solid laccase-producing medium contains 20.0g of malt extract, 10.0g of glucose and 3.0g of KH₂PO₄And 18.0g agar (natural pH 5.2-5.4); (2) inoculating trametes hirsuta cake with diameter of 0.8cm into 150mL glass triangular flask containing 50mL liquid laccase-producing medium and 0.25-1.0mM bisphenol A (0.25, 0.5 and 1.0mM bisphenol A) with a puncher, and keeping at 30 deg.C and 150 r.min in dark^-1Incubating for 7d by a water bath shaking table; sampling at regular time, measuring trametes hirsuta biomass, laccase activity, bisphenol A biotransformation rate, thallus adsorption amount and residual concentration in the culture solution, and calculating the biotransformation, adsorption and residual percentage of bisphenol A. High performance liquid chromatography (HPLC, Milford, MA, USA) for the detection of bisphenol a parameter settings: the mobile phase is 7:3(v/v) acetonitrile and water, the sample injection volume is 20 mu L, and the flow rate is 1.0 mL/min^-1And the ultraviolet wavelength is 278 nm.

As shown in FIG. 1, (a) the biotransformation efficiency of bisphenol A is 0.25mM, (b) the biotransformation efficiency of bisphenol A is 0.5mM, (c) the biotransformation efficiency of bisphenol A is 1.0mM, and the biotransformation efficiency of bisphenol A in the culture solution is as high as 100% after the culture solution is inoculated with trametes hirsuta (T.hirsute La-7) and cultured for 6d when the concentration of bisphenol A is 0.25-0.5 mM; while the highest adsorption amounts of the mycelia to bisphenol A were 5.9 and 14.1. mu. mol. g, respectively^-1(only 0.3% and 0.2% of the total bisphenol A, negligible) and adsorption occurred mainly in the first 3 d. Along with the extension of the inoculation time, the biomass of the trametes hirsuta is gradually increased, and the activity of the secreted extracellular laccase is increased and then decreased. This is because the secondary metabolites produced during the growth and reproduction of trametes hirsuta inhibit the extracellular laccase of myceliumYield and activity. When the concentration of bisphenol A is increased to 1.0mM, the biotransformation rate of bisphenol A in 7d of trametes hirsuta culture is low, the growth of mycelium is completely inhibited, and the activity of extracellular laccase is only 0.07 U.mL at most^-1. It can be seen that low doses of bisphenol A are efficiently converted by laccase-producing fungi, whereas high doses of bisphenol A completely inhibit fungal growth. When trametes hirsuta is used for treating high-concentration bisphenol A polluted water, careful consideration is needed. Can one directly adopt trametes hirsuta crude laccase liquid and mycelium homogenate to eliminate bisphenol A and realize simple, rapid and efficient purification of polluted water? This idea is urgently needed to be confirmed.

EXAMPLE 2 preparation of trametes hirsuta laccase solution and hypha homogenate

Trametes hirsutella (T.hirsute La-7) crude laccase liquid and hypha homogenate are prepared by a large-scale liquid fermentation culture method. The detailed preparation process is shown in figure 2: (1) subpackaging 50mL of liquid laccase-producing medium in 150mL glass triangular flask, inoculating fresh trametes hirsuta fungus cake with diameter of 0.8cm, and culturing at 30 deg.C and 150 r.min in dark^-1Incubating for 4d (the activity of extracellular laccase reaches the maximum) by a water bath shaking table; (2) filtering the culture solution with 4 layers of gauze to obtain supernatant and mycelium; then, the obtained supernatant was placed at 4 ℃ and 10000 r.min^-1Centrifuging for 5min to obtain trametes hirsuta crude laccase liquid (natural pH of 4.8); (3) weighing the collected mycelia, placing in a homogenizer for scattering and grinding into uniform paste; (4) 0.5g, 1.0 g and 2.0g of the paste were weighed out separately, and 20mL of 3.0 g.L was added^-1KH of₂PO₄Solutions (pH 4.8) were prepared at concentrations of 0.025, 0.05 and 0.1 g.mL, respectively^-1Homogenizing trametes hirsuta mycelium. And (3) determining the laccase activity of the trametes hirsuta laccase crude laccase liquid and the hypha homogenate liquid by using a cuvette color development method. The reaction system contained 20. mu.L of crude laccase solution (or 0.1g of hyphal homogenate), 3.4mL of 10mM citrate-phosphate buffer (pH 3.8), and 1.0mM 2, 6-dimethoxyphenol. The absorbance value at 468nm of the enzymatic reaction solution, the enzyme activity (U.mL) was recorded every 30s^-1Or U.g^-1) Defined as the amount of laccase change that causes one unit per minute.

As shown in FIG. 2, Trametes hirsutella (Trametes hirsute La-7) cake with a diameter of 0.8cm was inoculated in the liquid for laccase productionAfter culturing in the medium for 4 days, the laccase activity of the extracellular laccase crude solution reaches the maximum value of 1.3 U.mL^-1. At this time, the laccase activity bound to the surface of the mycelia was 0.6 U.g^-1(the water content of the mycelia was 60.1% by dry weight). Grinding mycelium completely to obtain uniform mycelium homogenate with milk white color, and measuring laccase activity to be 1.3 U.g^-1(dry weight). The results show that the trametes hirsuta crude laccase liquid and the mycelium homogenate liquid have the advantages of simple preparation process, low production cost and high laccase activity, and are expected to be used for simple, rapid and efficient conversion of bisphenol A in water.

Example 3 bioconversion Effect of laccase crude and hyphal homogenate on bisphenol A

The biotransformation effect of tramete (T.hirsute La-7) laccase crude liquid and mycelium homogenate liquid on bisphenol A is discussed by using a batch equilibrium test method. (1) The reaction system of the trametes hirsuta crude laccase liquid is built in a 50mL brown cylindrical glass container, and each glass container is separately filled with 20mL crude laccase liquid which respectively comprises 0.25mM bisphenol A, 0.5mM bisphenol A and 1.0mM bisphenol A; at the same time, Mn was investigated²⁺Concentration (0-2.0 mM MnSO)₄And 1mM H₂O₂) Influence on the conversion of trametes hirsuta crude laccase liquid to 0.5mM bisphenol A; (2) the reaction system of the trametes hirsuta mycelium homogenate is similar to that of the crude laccase liquid, and 0.025, 0.05 and 0.1 g/mL of the reaction liquid are respectively added into 20mL of the reaction liquid^-1Hyphal homogenates, and 0.5mM bisphenol A. The parameters of the enzymatic reaction are set to pH 4.8, 30 ℃ and 150 r.min^-1Incubating in water bath for 6h in dark place. At predetermined time intervals, 0.5mL of the reaction was aspirated and 0.5mL of chromatographically pure methanol was added rapidly to quench the free radical reaction initiated by the laccase. The mixture was quantitatively analyzed by HPLC for the residual concentration of bisphenol A and the biotransformation efficiency of bisphenol A was calculated.

As shown in FIG. 3 (a), trametes hirsutus (T.hirsute La-7) crude laccase solution can rapidly convert 0.25-1.0mM bisphenol A, and the biotransformation rate of bisphenol A gradually increases with the increase of the enzymatic reaction time. After 6 hours of enzymatic reaction, the biotransformation rate of the crude laccase liquid to the bisphenol A is as high as 80.6-90.7%. Addition of Mn²⁺Can promote the biotransformation rate of the trametes hirsuta crude laccase liquid to bisphenol A, wherein the Mn of 0.5mM²⁺Is best enhanced(FIG. 3 (b)). This is probably due to the small amount of Mn-dependent peroxidase contained in the crude laccase liquid, which is also capable of biotransformation of bisphenol A. FIG. 4 shows the bioconversion effect of mycelium and hyphal homogenates of trametes hirsutus (T.hirsute La-7) on bisphenol A. As can be seen from FIG. 4, the biotransformation efficiency of bisphenol A is directly correlated to the reaction time, wherein the transformation rate of the mycelium to bisphenol A increases rapidly after 120min, while the transformation rate of the mycelium homogenate to bisphenol A is higher within 90 min. When the addition amount of mycelium and mycelium homogenate is 0.025 g/mL^-1Increased to 0.1 g/mL^-1In the case of the two, the biotransformation rates of bisphenol A in 6h were respectively increased from 21.5% to 51.0% and from 58.1% to 84.2%. These results demonstrate that a laccase solution and mycelium homogenate prepared from trametes hirsutus (T.hirsute La-7) can be used for simple, rapid and efficient conversion of bisphenol A in water. Therefore, the trametes hirsuta crude laccase liquid and the mycelium homogenate liquid are utilized to research and develop the novel microbial agent, and the novel microbial agent has wide application potential and practical value in the aspects of treating bisphenol A polluted water body, recycling and reusing resources and the like.

Example 4 analysis and characterization of bisphenol A conversion product

LTQ-Orbitrap high resolution Mass Spectrometry (HRMS, Thermo Fisher Scientific, Bremen, Germany) was used in combination¹³C stability isotope ratio method, identifying trametes hirsuta (T.hirsute La-7) crude laccase liquid and mycelium homogenate liquid to convert bisphenol A intermediate product. The reaction system contained 10mL of crude laccase solution (or 10mL of 0.5g hyphal homogenate), 0.25mM bisphenol A, and 0.25mM¹³C₁₂Bisphenol A (molar ratio of bisphenol A monoisotope to isotopic label 1: 1). After the trametes hirsuta crude laccase liquid and the mycelium homogenate react for 2 and 5 hours respectively, 10mL of ethyl acetate is quickly added for stopping the enzymatic reaction, and an intermediate product is extracted. The mixed solution is shaken up by hand, and is kept stand for 6 hours after ultrasonic oscillation is carried out for 0.5 hour (repeated for 2 times) so that the ethyl acetate and the reaction solution are completely layered. Separating the ethyl acetate solution on the upper layer, concentrating and drying by nitrogen blowing, adding 1.0mL of chromatographic pure methanol to constant volume, and passing through a 0.22 mu m microporous filter membrane for detection. HRMS binding¹³The theoretical basis for identifying bisphenol A conversion intermediate products by using the C-stable isotope ratio mainly comprises the following three points: (1) the precise mass of the molecules is determined,the m/z of the product measured by HRMS can be accurate to four decimal places; (2) the difference between the isotopic label and the molecular mass of the product of the monoisotopic label and the isotopic label is 1.0033n (n represents¹³Number of C markers); (3) the relative intensity ratio of the product, the theoretical relative intensity ratio of the product of the single isotope and the isotope label is 1:1, 1:2:1 or 1:3:3:1, etc.

Trametes hirsuta (T.hirsute La-7) crude laccase liquid and hypha homogenate liquid can not only induce bisphenol A to be oxidized and cracked to generate micromolecule decomposition products, but also can induce bisphenol A free radicals to be covalently combined to form macromolecule polymerization products. Binding by HRMS, as shown in Table 1¹³C-stable isotope ratio method, three bisphenol A conversion intermediates were detected together in crude laccase liquid-started reaction system, and their m/z were 453.2064, 679.3059 and 133.0655, respectively. The products are preliminarily identified as bisphenol A dimer, trimer and 4-isopropyl phenol by an Xcalibur software analysis method, and the error range of the molecular mass is-1.2 to 1.3ppm (<5.0 ppm). Similarly, the presence of these products was also detected in the hyphal homogenate-mediated bisphenol a conversion system. The resulting oxidative decomposition of bisphenol A and free radical combination products significantly reduce the biological toxicity of the parent compound. In addition, the product molecular mass differences for the bisphenol a monoisotope and the isotopic label were 12.0395, 24.0794, 36.1200, and 6.0203 (theoretical values of 12.0400, 24.0800, 36.1200, and 6.0200, respectively), and the relative intensity ratios were 1.0:2.1:1.1, 1.0:3.0:2.9:1.0, and 1.0:0.9 (theoretical values of 1:2:1, 1:3:3:1, and 1:1, respectively). These results are corroborated and together confirm the accuracy and reliability of the presumed bisphenol a conversion products such as dimers, trimers and 4-isopropylphenol. Compared with the traditional method for identifying the product by mass spectrometry, HRMS is combined¹³The C-stability isotope ratio technology has the advantages of high detection accuracy, multiple cross validation and the like, and can quickly and accurately identify the conversion product of the bisphenol A in the complex environment matrix.

TABLE 1HRMS binding¹³C-tag identification of bisphenol A conversion intermediates

Claims

1. A method for rapidly converting bisphenol A in a water body is characterized in that the method comprises the steps of adding a water body containing bisphenol A with a preservation number of CGMCC No: 5.2199 fermented crude laccase liquid or mycelium homogenate of Trametes hirsute La-7.

2. The method of claim 1, wherein Mn is added during the addition of the fermentation laccase solution²⁺。

3. The method of claim 2, wherein Mn is Mn²⁺The concentration was 0.5 mM.

4. A method for the rapid conversion of bisphenol a in an aqueous body according to any of claims 1-3, wherein said fermented laccase liquid and mycelium homogenate are prepared by the following method: the preservation number is CGMCC No: 5.2199 trametes hirsutella (T. hirsute La-7) is inoculated into liquid medium, and the mixture is protected from light at 28-32 deg.C for 180 r.min^-1Fermenting and incubating for 3-6d, performing solid-liquid separation, wherein the supernatant is fermented crude laccase liquid, and mycelium is uniformly ground to obtain mycelium homogenate.

5. The method of claim 4, wherein the fermentation incubation parameters are 30 ℃ and 150 r-min^-1Liquid fermentation incubation 4 d.